The high proliferation rate of malignant cells is often associated with an increase in the rates of protein folding, assembly, and transport. The harsh tumor microenvironment also contributes to hyperactivation of the endoplasmic reticulum (ER) stress response as an important survival mechanism. The functional role of the ER stress response in mature B-cell leukemia or lymphoma has been largely overlooked because leukemia and lymphoma cells do not expand their ER like that of multiple myeloma cells. However, our studies have shown that chronic lymphocytic leukemia (CLL) indeed requires activation of the ER stress response pathway for survival. The IRE-1/XBP-1 pathway represents the most conserved ER stress response pathway. IRE-1 contains a luminal stress-sensor domain, and a cytoplasmic kinase/RNase domain. The RNase domain is critical for the function of IRE-1, because it splices 26 nucleotides from the XBP-1 mRNA, causing a frame shift in translation. The spliced XBP-1 mRNA encodes a functional 54-kDa XBP-1s transcription factor, which translocates to the nucleus and induces the expression of chaperones and lipids to aid in protein folding and trafficking. While most transcription factors remain undruggable, the specific activation mechanism of XBP-1 renders IRE-1 an attractive target for therapeutic intervention. We recently developed a structurally novel IRE-1 inhibitor, B-I09, and demonstrated its ability to block XBP- 1 expression in intact cells and to reduce CLL tumor burden in vivo. Genetic and pharmacological inhibition of the IRE-1/XBP-1 pathway compromises BCR signaling, and B-I09 synergizes with the BTK inhibitor ibrutinib to inhibit the growth of primary mouse and human B-cell cancer cells. This project seeks to utilize B-I09 and selected prodrug analogs with improved pharmacokinetic properties in combination with inhibitors of BCR signaling for the treatment of B-cell cancer. We have implemented an integrated approach encompassing mechanistic cell biology, in vivo efficacy studies, and chemical optimization to develop novel treatment strategies for CLL and other B-cell malignancies. In Aim 1, we proposed to evaluate the mechanism of action and synergy of B-I09 in combination with the BCR signaling pathway inhibitors ibrutinib (approved, mantle cell lymphoma, CLL) and fostamatinib (phase II, lymphoma) in B-cell cancer cell lines, primary mouse CLL cells, primary human samples, and in an in vivo E-TLC1 mouse model of CLL. In Aim 2, we will optimize the pharmacokinetic properties of inhibitors of XBP-1s expression and employ novel prodrug strategies to improve the bioavailability of this new class of anticancer agents for advanced preclinical studies.